Project Summary Cell fate specification is driven by lineage, signaling, and stochastic regulatory inputs. The mechanisms controlling stochastic fate specification, in which a cell randomly chooses between two or more fates, are poorly understood. Stochastic fate specification is critical for diversifying retinal neurons, olfactory sensory neurons, motor neurons, immune cells, and stem cells. Breakdowns in these mechanisms cause debilitating human disorders, including vision impairments, anosmia, autism, immunodeficiencies, and lymphoma. The main goal of this project is to determine how chromatin state and transcriptional variability control stochastic fate specification, using the random patterning of photoreceptor subtypes in the fly retina as a paradigm. The fly eye contains a random mosaic of two color-detecting R7 photoreceptor subtypes, defined by expression of Rhodopsin 4 (Rh4) or Rhodopsin 3 (Rh3). This fate decision is controlled by the transcription factor Spineless (Ss), which is expressed in a random subset of mature R7s. SsON R7s express Rh4, while SsOFF R7s express Rh3. Our data support a two-step mechanism regulating ssON/OFF expression in mature R7s. In step 1, the early enhancer drives an early pulse of ss transcription in R7 precursors that opens the chromatin at the ss locus. In step 2, the transcriptional pulse ceases and chromatin variably closes, defining the accessibility of the late enhancer. Depending on the degree of chromatin compaction, the late enhancer either turns on (open chromatin) or remains off (closed chromatin) for the lifetime of the mature R7. How regulation of transcription and chromatin compaction is integrated to turn genes randomly on or off during development is poorly understood. We will use DNA FISH, genomics, CRISPR, and lacO/LacI-based live imaging approaches to assess the role of chromatin dynamics and the temporality of regulatory inputs in the two-step mechanism (Aim 1). Identification of the source of variability driving stochastic fate specification in metazoans has not been achieved. Our data suggest that variability in transcription (initiation, elongation, frequency, and duration) in individual cells influences terminal R7 fate specification. To test this hypothesis, we will use nascent multi-color RNA FISH and MS2/MCP-based live imaging to assess transcriptional parameters and relate them to R7 subtype fates (Aim 2). Successful completion of these experiments will identify a source of variability that drives a cell fate decision and inform how molecular noise is utilized to diversify cell types during metazoan development.